Silk-based drug delivery system

ABSTRACT

The present invention provides for novel sustained release silk-based delivery systems. The invention further provides methods for producing such formulations. In general, a silk fibroin solution is combined with a therapeutic agent to form a silk fibroin article. The article is then treated in such a way as to alter its conformation. The change in conformation increases its crystallinity or liquid crystallinity, thus controlling the release of a therapeutic agent from the formulation. This can be accomplished as single material carriers or in a layer-by-layer fashion to load different therapeutic agents or different concentrations of these agents in each layer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S. patentapplication Ser. No. 13/783,485 filed on Mar. 4, 2013, which is acontinuation application of U.S. patent application Ser. No. 13/443,264filed on Apr. 10, 2012 and issued as U.S. Pat. No. 8,530,625 on Sep. 10,2013, which is a continuation application of U.S. patent applicationSer. No. 11/628,930 filed on Oct. 23, 2007 and issued as U.S. Pat. No.8,178,656 on May 15, 2012, which is a 35 U.S.C. § 371 U.S. NationalStage Entry of International Application No. PCT/US2005/020844 filed onJun. 13, 2005, which designates the U.S., and which claims the benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/579,065filed on Jun. 11, 2004, the contents of each of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to a silk-based drug deliverysystem. In particular, the system is capable of releasing a therapeuticagent from the device at a sustained and controllable rate.

BACKGROUND OF THE INVENTION

Silk, as the term is generally known in the art, means a filamentousfiber product secreted by an organism such as a silkworm or spider.Silks produced from insects, namely (i) Bombyx mori silkworms, and (ii)the glands of spiders, typically Nephilia clavipes, are the most oftenstudied forms of the material; however, hundreds to thousands of naturalvariants of silk exist in nature. Fibroin is produced and secreted by asilkworm's two silk glands.

Silkworm silk has been used in biomedical applications for over 1,000years. The Bombyx mori specie of silkworm produces a silk fiber (knownas a “bave”) and uses the fiber to build its cocoon. The bave, asproduced, includes two fibroin filaments or “broins”, which aresurrounded with a coating of gum, known as sericin—the silk fibroinfilament possesses significant mechanical integrity. When silk fibersare harvested for producing yarns or textiles, including sutures, aplurality of fibers can be aligned together, and the sericin ispartially dissolved and then resolidified to create a larger silk fiberstructure having more than two broins mutually embedded in a sericincoating.

The unique mechanical properties of reprocessed silk such as fibroin andits biocompatibility make the silk fibers especially attractive for usein biotechnological materials and medical applications. Silk provides animportant set of material options for biomaterials and tissueengineering because of the impressive mechanical properties,biocompatibility and biodegradability (Altman, G. H., et al.,Biomaterials 2003, 24, 401-416; Cappello, J., et al., J. Control.Release 1998, 53, 105-117; Foo, C. W. P., et al., Adv. Drug Deliver.Rev. 2002, 54, 1131-1143; Dinerman, A. A., et al., J. Control. Release2002, 82, 277-287; Megeed, Z., et al., Adv. Drug Deliver. Rev. 2002, 54,1075-1091; Petrini, P., et al., J. Mater. Sci-Mater. M. 2001, 12,849-853; Altman, G. H., et al., Biomaterials 2002, 23, 4131-4141;Panilaitis, B., et al., Biomaterials 2003, 24, 3079-3085). For example,3-dimensional porous silk scaffolds have been described for use intissue engineering (Meinel et al., Ann Biomed Eng. 2004 January;32(1):112-22; Nazarov, R., et al., Biomacromolecules in press). Further,regenerated silk fibroin films have been explored as oxygen- anddrug-permeable membranes, supports for enzyme immobilization, andsubstrates for cell culture (Minoura, N., et al., Polymer 1990, 31,265-269; Chen, J., et al., Minoura, N., Tanioka, A. 1994, 35, 2853-2856;Tsukada, M., et al., Polym. Sci. Part B Polym. Physics 1994, 32,961-968).

The desirability of sustained release has long been recognized in thepharmaceutical field. Sustained-release drug-delivery systems canprovide many benefits over conventional dosage forms. Generally,sustained-release preparations provide a longer period of therapeutic orprophylactic response compared to conventional rapid release dosageforms. For example, in treatment of pain, sustained-release formulationsare useful to maintain relatively constant analgesic drug release ratesover a period of time, for example 12-24 hours, so that blood serumconcentration of the drug remains at a therapeutically effective levelfor a longer duration than is possible with a conventional dosage formof the drug. In addition, whereas standard dosage forms typicallyexhibit high initial drug release rates that can result in unnecessarilyelevated blood serum levels of the drug, sustained-release formulationscan help maintain blood serum levels of the drug at or slightly abovethe therapeutically effective threshold. Such reduced fluctuation inblood serum concentration of the drug can also help prevent excessdosing.

Furthermore, sustained-release compositions, by optimizing the kineticsof delivery, also increase patient compliance as patients are lesslikely to miss a dose with less frequent administration, particularlywhen a once-a-day dosage regimen is possible; less frequentadministration also increases patient convenience. Sustained-releaseformulations may also reduce overall healthcare costs. Although theinitial cost of sustained-release delivery systems may be greater thanthe costs associated with conventional delivery systems, average costsof extended treatment over time can be lower due to less frequentdosing, enhanced therapeutic benefit, reduced side-effects, and areduction in the time required to dispense and administer the drug andmonitor patient compliance.

Many polymer-based systems have been proposed to accomplish the goal ofsustained release. These systems generally have relied upon eitherdegradation of the polymer or diffusion through the polymer as a meansto control release.

Polymer-based attempts to develop sustained-release formulations haveincluded the use of a variety of biodegradable and non-biodegradablepolymer (e.g. poly(lactide-co-glycolide)) microparticles containing theactive ingredient (see e.g., Wise et al., Contracgption, 1:227-234(1973); and Hutchinson et al., Biochem. Soc. Trans., 13:520-523 (1985)),and a variety of techniques are known by which active agents, e.g.proteins, can be incorporated into polymeric microspheres (see e.g.,U.S. Pat. No. 4,675,189 and references cited therein). In addition,various microcapsules, microparticles, and larger sustained-releaseimplants have been used to deliver pharmaceuticals to patients over anextended period of time. For example, polyesters such as poly-DL-lacticacid, polyglycolic acid, polylactide, and other copolymers, have beenused to release biologically active molecules such as progesterone andluteinizing hormone-releasing hormone (LH-RH) analogs, e.g., asdescribed in Kent et al., U.S. Pat. No. 4,675, 189, and Hutchinson etal., U.S. Pat. No. 4,767,628.

Unfortunately, the successes of current polymer-based sustained deliverysystems have been limited. This is due, in large part, to theirnecessity on using organic solvents during preparation. Even solventswhich are well tolerated in vivo, i.e. ethylacetate, may causeimmunological reactions or anaphylactic shock. In addition, all organicsolvents are volatile and require expensive production processes.

There is, therefore, a need for a biocompatible, biodegradable,sustained-release drug-delivery system. Such products should have thedesired mechanical properties of tensile strength, elasticity,formability, and the like, provide for controlled resorption, and bephysiologically acceptable. Moreover, such products should allow forease of administration for a variety of in vivo indications and inbest-case scenarios be inexpensive to manufacture.

SUMMARY OF THE INVENTION

The present invention provides a novel sustained release silk-based drugdelivery system. The invention further provides methods for producingsuch devices.

In one embodiment, a method for producing a pharmaceutical formulationfor controlled release of a therapeutic agent is provided. The methodcomprises contacting a silk fibroin solution with the therapeutic agent.Therapeutic agents include, for example, proteins, peptides and smallmolecules. In a preferred embodiment, an aqueous silk fibroin solutionis utilized.

Next, a silk fibroin article that contains the therapeutic agent isformed. The silk fibroin article may be a thread, fiber, film, foam,mesh, hydrogel, three-dimensional scaffold, tablet filling material,tablet coating, or microsphere.

The conformation of the article is then altered in order to increase itscrystallinity or liquid crystlallinity, thus providing controlledrelease of the therapeutic agent from the silk fibroin article.

In one embodiment of the present invention, the conformation of thearticle is altered by contacting the fibroin article with methanol. Themethanol concentration is at least 50%, at least 70%, at least 90% or atleast 100%.

In an alternative embodiment, alteration in the conformation of thefibroin article is induced by treating the article with sheer stress.The sheer stress may be applied by passing the article through a needle.

The conformation of the fibroin article may also be altered bycontacting the article with an electric field, by applying pressure, orby contacting the article with salt.

Preferably, the therapeutic agent is equal to or greater than about 10kilodaltons (kDa). More preferably the therapeutic agent is greater thanabout 20 kDa.

In a further embodiment, a pharmaceutical formulation with a pluralityof silk fibroin articles (i.e. layers) is provided. In this embodiment,at least one layer has an induced conformational change that differsfrom at least one other layer. The silk fibroin article layers may eachcontain different therapeutic agents, each layer having the same ordifferent induced conformational changes.

The pharmaceutical formulation is biodegradable and may comprise atargeting agent that specifically targets the device to a specific cellor tissue type. The targeting agent may be, for example, a sugar,peptide, or fatty acid.

In one embodiment, the silk fibroin solution is obtained from a solutioncontaining a dissolved silkworm silk, such as, for example, from Bombyxmori. Alternatively, the silk fibroin solution is obtained from asolution containing a dissolved spider silk, such as, for example, fromNephila clavipes. The silk fibroin solution may also be obtained from asolution containing a genetically engineered silk. In one embodiment,the genetically engineered silk comprises a therapeutic agent. This maybe a fusion protein with a cytokine, an enzyme, or any number ofhormones or peptide-based drugs, antimicrobials and related substrates.

Also encompassed in the present invention is the pharmaceuticalformulation for controlled release of a therapeutic agent, produced bythe above methods.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the invention and,together with the description, serve to explain the objects, advantages,and principles of the invention.

FIG. 1 shows the drug release of FD4 after 24, 48, and 130 hours.

FIG. 2 shows the influence of methanol concentration on drug release.Treatment with methanol concentrations up to 50% results in a high burstrelease within the first 12 hours with minute amounts of FD4 released atlater time points. In contrast, treatment with 90% or 100% methanolsolutions results in a sustained and faster release for about 200 hours.

FIG. 3 shows coating of core with silk fibroin solution treated withdifferent concentrations of methanol (I) core without coating; (d) corewith coating treated with 90% methanol solution; (c) core with twolayers of fibroin; and (b) core with three layers of fibroin. Theresults demonstrate that the release of a therapeutic can be controlledthrough the thickness of the coating around the core.

FIG. 4 shows release of FITC-dextrans with different molecular weightsand from silk films treated with H2O (4A) or methanol (4B).

FIG. 5 shows cumulative release and adsorption of horseradish peroxidase(HRP; 5A, 5C) and Lysozyme (Lys, 5B, 5D) from silk films treated withmethanol or H2O, respectively.

FIG. 6 shows AFM images of native silk films (6A, 6C) or films treatedwith methanol (6B, 6D). Bar length 2.5 μm (6A, 6B) and 0.5 μm (6B, 6D).

FIG. 7 shows physicochemical characterization of native silk films orfilms treated with methanol. (7A) FTIR analysis and X-ray diffractogrammof methanol treated (7B) and untreated (7C) films. Contact anglemeasurements of a water drop on methanol treated (7D) and untreated (7E)films over time.

DETAILED DESCRIPTION OF THE INVENTION

Methods for preparation of silk-based drug delivery systems aredescribed. In particular, the drug delivery system allows for thecontrolled and sustained release of therapeutic agents in vivo. Ingeneral, a silk fibroin solution is combined with a therapeutic agent toform a silk fibroin article. The article is then treated in such a wayas to alter its conformation. The change in conformation increases itscrytallinity, thus controlling the release of a therapeutic agent fromthe formulation.

As used herein, the term “fibroin” includes silkworm fibroin and insector spider silk protein (Lucas et al., Adv. Protein Chem 13: 107-242(1958)). Preferably, fibroin is obtained from a solution containing adissolved silkworm silk or spider silk. The silkworm silk protein isobtained, for example, from Bombyx mori, and the spider silk is obtainedfrom Nephila clavipes. In the alternative, the silk proteins suitablefor use in the present invention can be obtained from a solutioncontaining a genetically engineered silk, such as from bacteria, yeast,mammalian cells, transgenic animals or transgenic plants. See, forexample, WO 97/08315 and U.S. Pat. No. 5,245,012.

The silk fibroin solution can be prepared by any conventional methodknown to one skilled in the art. For example, B. mori cocoons are boiledfor about 30 minutes in an aqueous solution. Preferably, the aqueoussolution is about 0.02M Na₂CO₃. The cocoons are rinsed, for example,with water to extract the sericin proteins and the extracted silk isdissolved in an aqueous salt solution. Salts useful for this purposeinclude lithium bromide, lithium thiocyanate, calcium nitrate or otherchemicals capable of solubilizing silk. Preferably, the extracted silkis dissolved in about 9-12 M LiBr solution. The salt is consequentlyremoved using, for example, dialysis.

If necessary, the solution can then be concentrated using, for example,dialysis against a hygroscopic polymer, for example, PEG, a polyethyleneoxide, amylose or sericin.

Preferably, the PEG is of a molecular weight of 8,000-10,000 g/mol andhas a concentration of 25-50%. A slide-a-lyzer dialysis cassette(Pierce, MW CO 3500) is preferably used. However, any dialysis systemmay be used. The dialysis is for a time period sufficient to result in afinal concentration of aqueous silk solution between 10-30%. In mostcases dialysis for 2-12 hours is sufficient. See, for example, PCTapplication PCT/US/04/11199.

Alternatively, the silk fibroin solution can be produced using organicsolvents. Such methods have been described, for example, in Li, M., etal., J. Appl. Poly Sci. 2001, 79, 2192-2199; Min, S., et al. Sen'IGakkaishi 1997, 54, 85-92; Nazarov, R. et al., Biomacromolecules 2004May-June; 5(3):718-26.

In accordance with the present invention, the silk fibroin solutionscontain at least one therapeutic agent. The silk fibroin solution iscontacted with a therapeutic agent prior to forming the fibroin article,e.g. a fiber, mesh, scaffold, or loaded into the article after it isformed. For loading after formation, silk assembly is used to controlhydrophilic/hydrophobic partitioning (see, for example, Jin et al.,Nature. 2003 Aug. 28; 424(6952):1057-61) and the adsorption of phaseseparation of the therapeutic agent. The material can also be loaded byentrapping the therapeutic agent in the silk by inducing the transitionto the beta sheet (e.g. methanol, shear, salts, electric) and addinglayers on this with each layer entrapping the next therapeutic. Thislayer-by-layer approach would allow onion like structures with selectiveloading in each layer.

The variety of different therapeutic agents that can be used inconjunction with the formulations of the present invention is vast andincludes small molecules, proteins, peptides and nucleic acids. Ingeneral, therapeutic agents which may be administered via the inventioninclude, without limitation: anti-infectives such as antibiotics andantiviral agents; chemotherapeutic agents (i.e. anticancer agents);anti-rejection agents; analgesics and analgesic combinations;anti-inflammatory agents; hormones such as steroids; growth factors(bone morphogenic proteins (i.e. BMP's 1-7), bone morphogenic-likeproteins (i.e. GFD-5, GFD-7 and GFD-8), epidermal growth factor (EGF),fibroblast growth factor (i.e. FGF 1-9), platelet derived growth factor(PDGF), insulin like growth factor (IGF-I and IGF-II), transforminggrowth factors (i.e. TGF-β-III), vascular endothelial growth factor(VEGF)); anti-angiogenic proteins such as endostatin, and othernaturally derived or genetically engineered proteins, polysaccharides,glycoproteins, or lipoproteins. Growth factors are described in TheCellular and Molecular Basis of Bone Formation and Repair by Vicki Rosenand R. Scott Thies, published by R. G. Landes Company, herebyincorporated herein by reference.

Additionally, the silk based devices of the present invention can beused to deliver any type of molecular compound, such as, pharmacologicalmaterials, vitamins, sedatives, steroids, hypnotics, antibiotics,chemotherapeutic agents, prostaglandins, and radiopharmaceuticals. Thedelivery system of the present invention is suitable for delivery of theabove materials and others including but not limited to proteins,peptides, nucleotides, carbohydrates, simple sugars, cells, genes,anti-thrombotics, anti-metabolics, growth factor inhibitor, growthpromoters, anticoagulants, antimitotics, fibrinolytics,anti-inflammatory steroids, and monoclonal antibodies.

Additionally, the pharmaceutical formulation of the present inventionmay also have a targeting ligand. Targeting ligand refers to anymaterial or substance which may promote targeting of the pharmaceuticalformulation to tissues and/or receptors in vivo and/or in vitro with theformulations of the present invention. The targeting ligand may besynthetic, semi-synthetic, or naturally-occurring. Materials orsubstances which may serve as targeting ligands include, for example,proteins, including antibodies, antibody fragments, hormones, hormoneanalogues, glycoproteins and lectins, peptides, polypeptides, aminoacids, sugars, saccharides, including monosaccharides andpolysaccharides, carbohydrates, vitamins, steroids, steroid analogs,hormones, cofactors, and genetic material, including nucleosides,nucleotides, nucleotide acid constructs, petptide nucleic acids (PNA),aptamers, and polynucleotides. Other targeting ligands in the presentinvention include cell adhesion molecules (CAM), among which are, forexample, cytokines, integrins, cadherins, immunoglobulins and selectin.The pharmaceutical formulations of the present invention may alsoencompass precursor targeting ligands. A precursor to a targeting ligandrefers to any material or substance which may be converted to atargeting ligand. Such conversion may involve, for example, anchoring aprecursor to a targeting ligand. Exemplary targeting precursor moietiesinclude maleimide groups, disulfide groups, such as ortho-pyridyldisulfide, vinylsulfone groups, azide groups, and [agr]-iodo acetylgroups.

Silk formulations containing bioactive materials may be formulated bymixing one or more therapeutic agents with the silk solution used tomake the article. Alternatively, a therapeutic agent can be coated ontothe pre-formed silk fibroin article, preferably with a pharmaceuticallyacceptable carrier. Any pharmaceutical carrier can be used that does notdissolve the silk material. The therapeutic agents may be present as aliquid, a finely divided solid, or any other appropriate physical form.

The above described silk fibroin solution, which contains at least onetherapeutic agent, is next processed into a thread, fiber, film, mesh,hydrogel, three-dimensional scaffold, tablet filling material, tabletcoating, or microsphere. Methods for generating such are well known inthe art. See, e.g. Altman, et al., Biomaterials 24:401, 2003; PCTPublications, WO 2004/000915 and WO 2004/001103; and PCT ApplicationNo's PCT/US/04/11199 and PCT/U504/00255, which are herein incorporatedby reference.

Silk films can be produced by preparing the concentrated aqueous silkfibroin solution and casting the solution. See, for example PCTapplication PCT/US/04/11199. The film can be contacted with water orwater vapor, in the absence of alcohol. The film can then be drawn orstretched mono-axially or biaxially. The stretching of a silk blend filminduces molecular alignment of the film and thereby improves themechanical properties of the film.

If desired, the film comprises from about 50 to about 99.99 part byvolume aqueous silk protein solution and from about 0.01 to about 50part by volume biocompatible polymer e.g., polyethylene oxide (PEO).Preferably, the resulting silk blend film is from about 60 to about 240μm thick, however, thicker samples can easily be formed by using largervolumes or by depositing multiple layers.

Foams may be made from methods known in the art, including, for example,freeze—drying and gas foaming in which water is the solvent or nitrogenor other gas is the blowing agent, respectively. Alternately, the foamis made by contacting the silk fibroin solution with granular salt. Thepore size of foams can be controlled, for example by adjusting theconcentration of silk fibroin and the particle size of a granular salt(for example, the preferred diameter of the salt particle is betweenabout 50 microns and about 1000 microns). The salts can be monovalent ordivalent. Preferred salts are monovalent, such as NaCl and KCl. Divalentsalts, such as CaCl₂ can also be used. Contacting the concentrated silkfibroin solution with salt is sufficient to induce a conformationalchange of the amorphous silk to a β-sheet structure that is insoluble inthe solution. After formation of the foam, the excess salt is thenextracted, for example, by immersing in water. The resultant porous foamcan then be dried and the foam can be used, for example, as a cellscaffold in biomedical application. See, for example PCT applicationPCT/US/04/11199.

In one embodiment, the foam is micropatterned foam. Micropatterned foamscan be prepared using, for example, the method set forth in U.S. Pat.No. 6,423,252, the disclosure of which is incorporated herein byreference. The method comprises contacting the concentrated silksolution with a surface of a mold, the mold comprising on at least onesurface thereof a three-dimensional negative configuration of apredetermined micropattern to be disposed on and integral with at leastone surface of the foam, lyophilizing the solution while in contact withthe micropatterned surface of the mold, thereby providing a lyophilized,micropatterned foam, and removing the lyophilized, micropatterned foamfrom the mold. Foams prepared according to this method comprise apredetermined and designed micropattern on at least one surface, whichpattern is effective to facilitate tissue repair, ingrowth orregeneration.

Fibers may be produced using, for example, wet spinning orelectrospinning Alternatively, as the concentrated solution has agel-like consistency, a fiber can be pulled directly from the solution.

Electrospinning can be performed by any means known in the art (see, forexample, U.S. Pat. No. 6,110,590). Preferably, a steel capillary tubewith a 1.0 mm internal diameter tip is mounted on an adjustable,electrically insulated stand. Preferably, the capillary tube ismaintained at a high electric potential and mounted in the parallelplate geometry. The capillary tube is preferably connected to a syringefilled with silk solution. Preferably, a constant volume flow rate ismaintained using a syringe pump, set to keep the solution at the tip ofthe tube without dripping. The electric potential, solution flow rate,and the distance between the capillary tip and the collection screen areadjusted so that a stable jet is obtained. Dry or wet fibers arecollected by varying the distance between the capillary tip and thecollection screen.

A collection screen suitable for collecting silk fibers can be a wiremesh, a polymeric mesh, or a water bath. Alternatively and preferably,the collection screen is an aluminum foil. The aluminum foil can becoated with Teflon fluid to make peeling off the silk fibers easier. Oneskilled in the art will be able to readily select other means ofcollecting the fiber solution as it travels through the electric field.The electric potential difference between the capillary tip and thealuminum foil counter electrode is, preferably, gradually increased toabout 12 kV, however, one skilled in the art should be able to adjustthe electric potential to achieve suitable jet stream.

The present invention additionally provides a non-woven network offibers comprising a pharmaceutical formulation of the present invention.The fiber may also be formed into yarns and fabrics including forexample, woven or weaved fabrics.

The fibroin silk article of the present invention may also be coatedonto various shaped articles including biomedical devices (e.g. stents),and silk or other fibers, including fragments of such fibers.

Silk hydrogels can be prepared by methods known in the art, see forexample PCT application PCT/US/04/11199. The sol-gel transition of theconcentrated silk fibroin solution can be modified by changes in silkfibroin concentration, temperature, salt concentrations (e.g. CaCl2,NaCl, and KCl), pH, hydrophilic polymers, and the like. Before thesol-gel transition, the concentrated aqueous silk solution can be placedin a mold or form. The resulting hydrogel can then be cut into anyshape, using, for example a laser.

The silk fibroin articles described herein can be further modified afterfabrication. For example, the scaffolds can be coated with additives,such as bioactive substances that function as receptors orchemoattractors for a desired population of cells. The coating can beapplied through absorption or chemical bonding.

Additives suitable for use with the present invention includebiologically or pharmaceutically active compounds. Examples ofbiologically active compounds include, but are not limited to: cellattachment mediators, such as collagen, elastin, fibronectin,vitronectin, laminin, proteoglycans, or peptides containing knownintegrin binding domains e.g. “RGD” integrin binding sequence, orvariations thereof, that are known to affect cellular attachment(Schaffner P & Dard 2003 Cell Mol Life Sci. January; 60(1):119-32;Hersel U. et al. 2003 Biomaterials. November; 24(24):4385-415);biologically active ligands; and substances that enhance or excludeparticular varieties of cellular or tissue ingrowth. Other examples ofadditive agents that enhance proliferation or differentiation include,but are not limited to, osteoinductive substances, such as bonemorphogenic proteins (BMP); cytokines, growth factors such as epidermalgrowth factor (EGF), platelet-derived growth factor (PDGF), insulin-likegrowth factor (IGF-I and II) TGF-β, and the like. As used herein, theterm additive also encompasses antibodies, DNA, RNA, modifiedRNA/protein composites, glycogens or other sugars, and alcohols.

Biocompatible polymers can be added to the silk article to generatecomposite matrices in the process of the present invention.

Biocompatible polymers useful in the present invention include, forexample, polyethylene oxide (PEO) (U.S. Pat. No. 6,302,848),polyethylene glycol (PEG) (U.S. Pat. No. 6,395,734), collagen (U.S. Pat.No. 6,127,143), fibronectin (U.S. Pat. No. 5,263,992), keratin (U.S.Pat. No. 6,379,690), polyaspartic acid (U.S. Pat. No. 5,015,476),polylysine (U.S. Pat. No. 4,806,355), alginate (U.S. Pat. No.6,372,244), chitosan (U.S. Pat. No. 6,310,188), chitin (U.S. Pat. No.5,093,489), hyaluronic acid (U.S. Pat. No. 387,413), pectin (U.S. Pat.No. 6,325,810), polycaprolactone (U.S. Pat. No. 6,337,198), polylacticacid (U.S. Pat. No. 6,267,776), polyglycolic acid (U.S. Pat. No.5,576,881), polyhydroxyalkanoates (U.S. Pat. No. 6,245,537), dextrans(U.S. Pat. No. 5,902,800), and polyanhydrides (U.S. Pat. No. 5,270,419).Two or more biocompatible polymers can be used.

As a next step in the method for producing pharmaceutical formulationsfor controlled release of therapeutic agents, the conformation of thesilk fibroin article is altered. The induced conformational changealters the crystallinity of the article, thus altering the rate ofrelease of the therapeutic agent from the silk fibroin article. Theconformational change may be induced by treating the fibroin articlewith methanol. The methanol concentration is at least 50%, at least 70%,at least 90% or at least 100%.

Alternatively, the alteration in the conformation of the fibroin articlemay be induced by treating the article with sheer stress. The sheerstress may be applied, for example, by passing the article through aneedle. Other methods of inducing conformational changes includecontacting the article with an electric field, salt or by applyingpressure.

The silk-based drug delivery system of the present invention maycomprise a plurality (i.e. layers) of silk fibroin articles, where atleast one silk fibroin article may have an induced conformational changethat differs from at least one alternative silk fibroin article. Forexample, each layer may have different solutions of fibroin(concentrations, drugs) and have different conformational changes. Thesecan be combined in various sequences to create ‘onion-like’ structuressuch that the delivery vehicle will offer changing rates of release ofeach layer depending on crystallinity, thickness, concentration of drug,type of drug, etc. This approach is very amenable to scale up andcombinatorial or related approaches to formulation to create multiplecontrol points in release profiles and drug combinations.

Additionally, the release of the therapeutic agent from thepharmaceutical formulations of the present invention can be controlledthrough the thickness of the silk fibroin article. As shown in FIG. 3,with increasing article thickness, the initial burst and amount of drugreleased within the first 100 hours was reduced. However, the sustainedrelease of drug over time was significantly higher with increasing filmnumbers.

Drug delivery composites are also encompassed. A family of suchstructures are prepared as above and then dispersed in various amountsinto the fibroin hydrogels. These composite systems would then be usedin various modes of delivery, such as, for example, the “onion-like”vehicles described above.

The materials produced using the present invention, e.g., hydrogels,fibers, films, foams, or meshes, may be used in a variety of medicalapplications such as a drug (e.g, small molecule, protein, or nucleicacid) delivery device, including controlled release systems.

Controlled release permits dosages to be administered over time, withcontrolled release kinetics. In some instances, delivery of thetherapeutic agent is continuous to the site where treatment is needed,for example, over several weeks. Controlled release over time, forexample, over several days or weeks, or longer, permits continuousdelivery of the therapeutic agent to obtain optimal treatment. Thecontrolled delivery vehicle is advantageous because it protects thetherapeutic agent from degradation in vivo in body fluids and tissue,for example, by proteases.

Controlled release from the pharmaceutical formulation may be designedto occur over time, for example, for greater than about 12 or 24 hours.The time of release may be selected, for example, to occur over a timeperiod of about 12 hours to 24 hours; about 12 hours to 42 hours; or, e.g., about 12 to 72 hours. In another embodiment, release may occur forexample on the order of about 2 to 90 days, for example, about 3 to 60days. In one embodiment, the therapeutic agent is delivered locally overa time period of about 7-21 days, or about 3 to 10 days. In otherinstances, the therapeutic agent is administered over 1,2,3 or moreweeks in a controlled dosage. The controlled release time may beselected based on the condition treated. For example, longer times maybe more effective for wound healing, whereas shorter delivery times maybe more useful for some cardiovascular applications.

Controlled release of the therapeutic agent from the fibroin article invivo may occur, for example, in the amount of about 1 ng to 1 mg/day,for example, about 50 ng to 500 pg/day, or, in one embodiment, about 100ng/day. Delivery systems comprising therapeutic agent and a carrier maybe formulated that include, for example, 10 ng to 1 mg therapeuticagent, or in another embodiment, about 1 ug to 500 ug, or, for example,about 10 ug to 100 ug, depending on the therapeutic application.

The silk-based drug delivery vehicle may be administered by a variety ofroutes known in the art including topical, oral, parenteral (includingintravenous, intraperitoneal, intramuscular and subcutaneous injectionas well as intranasal or inhalation administration) and implantation.The delivery may be systemic, regional, or local. Additionally, thedelivery may be intrathecal, e. g., for CNS delivery. For example,administration of the pharmaceutical formulation for the treatment ofwounds may be by topical application, systemic administration by enteralor parenteral routes, or local or regional injection or implantation.The silk-based vehicle may be formulated into appropriate forms fordifferent routes of administration as described in the art, for example,in “Remington: The Science and Practice of Pharmacy”, Mack PublishingCompany, Pennsylvania, 1995, the disclosure of which is incorporatedherein by reference.

The controlled release vehicle may include excipients available in theart, such as diluents, solvents, buffers, solubilizers, suspendingagents, viscosity controlling agents, binders, lubricants, surfactants,preservatives and stabilizers. The formulations may include bulkingagents, chelating agents, and antioxidants. Where parenteralformulations are used, the formulation may additionally or alternatelyinclude sugars, amino acids, or electrolytes.

Excipients include polyols, for example of a molecular weight less thanabout 70,000 kD, such as trehalose, mannitol, and polyethylene glycol.See for example, U. S. Pat. No. 5,589,167, the disclosure of which isincorporated herein. Exemplary surfactants include nonionic surfactants,such as Tweeng surfactants, polysorbates, such as polysorbate 20 or 80,etc., and the poloxamers, such as poloxamer 184 or 188, Pluronic (r)polyols, and other ethylene/polypropylene block polymers, etc. Buffersinclude Tris, citrate, succinate, acetate, or histidine buffers.Preservatives include phenol, benzyl alcohol, metacresol, methylparaben, propyl paraben, benzalconium chloride, and benzethoniumchloride. Other additives include carboxymethylcellulose, dextran, andgelatin. Stabilizing agents include heparin, pentosan polysulfate andother heparinoids, and divalent cations such as magnesium and zinc.

The pharmaceutical formulation of the present invention may besterilized using conventional sterilization process such as radiationbased sterilization (i.e. gamma-ray), chemical based sterilization(ethylene oxide), autoclaving, or other appropriate procedures.Preferably the sterilization process will be with ethylene oxide at atemperature between 52-55° C. for a time of 8 or less hours. Aftersterilization the formulation may be packaged in an appropriatesterilize moisture resistant package for shipment.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of theinvention, the preferred methods and materials are described below. Allpublications, patent applications, patents and other referencesmentioned herein are incorporated by reference. In addition, thematerials, methods and examples are illustrative only and not intendedto be limiting. In case of conflict, the present specification,including definitions, controls.

The invention will be further characterized by the following exampleswhich are intended to be exemplary of the invention.

EXAMPLES Example I

Materials and Methods: Preparation of Silk

Silk cocoons were cut in quarters and washed in Na₂CO₃ solution for 1hour. The silk was washed 2 times with hot water and 20 times with coldwater. Silk was dried over night and dissolved in 9M LiBr to become a10% silk-fibroin solution. The solution was centrifuged at 27,000 g for30 minutes and the supernatant was transferred in a dialysis cassette(MWCO 3,500) and dialyzed for 2 days. Silk-fibroin concentration wasadjusted to 5% (m/V in water) by evaporation at 250 mbar and 45° C. Theresulting silk solution was either mixed with a solution of FITC coupledto dextran (molecular weight of 4 kDa; FD4; concentration 10 mg/ml,Sigma) in a ratio of 150 ul silk-fibroin solution to 20 ul of FD4solution, or the FD-4 was added later for control groups (see table 1).As a further group, parts of this solution were sonicated with 50 Hz,2A, 10 sec (Hielscher, UP200H). 170 ul of the solution (FD-4/silkmixture) was added into each well of a 96 well plate. For control, wellswere filled with 150 ul of the silk-fibroin solution. Water wasevaporated over night at room temperature and 250 mbar. 20 ul of the FD4solution was added to the wells, which received the silk-fibroinsolution only (to analyze the effect of mixing the drug with the solvedsilk-fibroin vs. later incubation of the solid films). Further wellswere not incubated with silk (to analyze a possible fluorescence of thesilk-fibroin films themselves). The films were either treated withwater, methanol 20% or methanol 90% (V/V) for 3 hours. The solutionswere aspirated and replaced by 300 ul PBS for the drug release study.Total release medium was replaced with fresh PBS after 24, 48, 130 hoursand fluorescence was read with a Lumicounter (Packard, 480V, Gain Level,1, ex. 485 nm, em. 530 nm).

Results & Discussion:

Plain silk-fibroin films do not show fluorescence. Without MeOHtreatment or treatment with 20% MeOH, the release was characterized by ahigh initial burst and only minute release after 24 hours (Groups I+0and I+20), although more FD4 was released when treated with 20% MeOH.This is probably due to the reduced solubility of FD4 in presence ofMeOH. When treated with 90% MeOH, significantly more FD4 was releasedafter 48 and after 130 hours, demonstrating the feasibility to get asustained release from silk polymers by inducing a transformationalchange (I+90). Similar to the observation with the groups I+0 and I+20,the increase in MeOH concentration to 90% results in a higherencapsulation of total FD4. An incubation of the prepared film with FD4for 3 hours (I−90) results in a high initial burst, similar to the I+0and I+20. The absence of a substantial sustained release is probably dueto a hindrance of drug diffusion in the (amorphous) silk-fibroin films.Therefore, conformational changes do not substantially affect FD4absorbed to the surface of the films. Essentially the same results wereobtained for the groups, which were also treated with ultrasonication.However, a treatment with ultrasonication does not have an influence ondrug release as compared to the non sonicated group.

In conclusion, a sustained release of drugs (FD4) can be obtained withsilk-fibroin polymers, when a conformational change is induced with 90%MeOH. Ultrasonication does not have an impact on drug release.

Example 2

We evaluated the feasibility to formulate drug delivery systems based onsilk-fibroin. The experiments started from an aqueous fibroin-drugsolution, followed by a slow evaporation of the water resulting in asolid fibroin film with suspended drug molecules. A conformationalchange of the fibroin was induced through methanol treatment, resultingin an increase of crystallinity. The degree of crystallinity governedthe release of the drug.

Results & Discussion

Dextrans coupled to a fluorescent dye were chosen as a model drug. Theyallow a straightforward assessment of the influence of drug molecularweight on swelling gels. FD4—a term used in this summary describes FITC(F) coupled to a dextran (D) with a molecular weight of 4,000 g/mol. Afirst set of experiments compared FD4 release from fibroin gels treatedwith water and ascending methanol concentrations (FIG. 2).

FIG. 2 shows the release of FD4 over time. The differences in maximumFD4 concentrations stems from different solubilities of the drug inmethanol solutions of different concentrations or water. However, therelease pattern was different. Treatment with methanol concentrations upto 50% resulted in a high burst release within the first 12 hours withminute amounts of FD4 released at later time points. In contrast,treatment with 90% or 100% methanol solutions resulted in a sustainedand faster release for about 200 hours. At later time-points, the amountof drug released per time (slope) significantly decreased, with therelease continuing throughout the observation period (exceeding 1,000hours). Treatment with shear stress alone (syringe treated, FIG. 2) alsoinduced gel formation through an increase of crystallinity.

To control the high initial burst -more than 30% of the total FD4 used-,identical films were prepared and treated with 90% methanol (core). Thiscore was coated with additional layers of fibroin and again treated with90% methanol solution or water (FIG. 3).

The results demonstrated in FIG. 3 show that the release can becontrolled through the thickness of the coating around the core. Withincreasing film thickness (d-b-c in FIG. 3), the initial burst wasreduced, the amount of drug released within the first 50 hourssignificantly less as compared to the core (I, FIG. 3), and the drugrelease (slope) was significantly higher for the coated cores than forthe uncoated one. In particular the cores coated with 2 (c) or 3 (b)layers of fibroin had a nearly linear release for the first 300 hours(about 12.5 days), following zero order kinetics. The coating had aminimal effect on drug release, when treated with water instead ofmethanol solution, demonstrating the importance of inducing theconformational change (data not shown).

These findings allow for at least 2 conclusions: (i) silk fibroin whentreated with methanol solution can be used to fabricate a controlleddrug delivery system; and (ii) silk-fibroin coatings, treated withmethanol solution can modify the release of drugs from a drug containingcore.

This allows for numerous applications, including the preparation ofmicrospheres or the coating of tablets to modify the release.

Example 3

The exposure of silk films to methanol suggested an increase incrystallinity, as determined by FTIR analysis (FIG. 7A). This findingwas based on a Amide II bond shift from 1540 cm-1 to 1535 cm-1, afinding typical for the increase in┌ β-crystalline structures.Similarly, an additional shoulder appeared in response to methanoltreatment at 1630 cm-1 (Amide I) and 1265 cm-1 (Amide III).

This data was corroborated by X-ray diffractometry (FIGS. 7B and 7C).The hydrophobicity of the film surfaces were significantly influenced bythe crystallinity change of the films in response to methanol treatment,as determined by contact angle measurements (FIGS. 7D and 7E). Formethanol treated films no change of the contact angle was observed overtime, indicating the water-insolubility of these films as opposed towater treated films, in which a rapid decrease in contact anglesresulted after 3 minutes of exposure to a water droplet.

The topology of silk films before and after methanol treatment wasassessed by atomic force microscopy (FIG. 6). Exposure to methanol asopposed to untreated films resulted in a rougher surface (FIGS. 6A and6B) and the formation of globular structures (FIGS. 6C and 6D).

Conclusion: Methanol treatment of silk films resulted in an increase incrystallinity (β-sheet), an increase in hydrophobicity, a decrease inwater solubility, and a change in surface topology.

Example 4

The release of fluorescently marked dextrans with different molecularweights was evaluated as a function of methanol treatment.

The release of dextrans with size ranges from 4 to 20 kDa was notapparently sustained, whereas the release of larger molecules (40 kDa)was retarded (FIG. 4A). In contrast, the methanol treatment of the silkfilms resulted in a strong retardation of release for all dextrans,particularly for molecular weights equal to or exceeding 10 kDa (FIG.4B).

The efficacy of silk films as drug delivery systems for protein drugswas evaluated using horseradish peroxidase (HRP) and Lysozyme (Lys; FIG.5), and analyzed by biological potency tests. A discontinuous releasefrom native silk films was observed for HRP, characterized by an initialburst of 5% of the total loading, followed by a lag phase of two daysand a continuous release from days 3 to 8 (FIG. 5A). HRP release wassignificantly changed after exposure of the HRP loaded films tomethanol. No initial burst was observed and the HRP release started atday 5, from which on it was continuously released until day 23 (FIG.5A). Lysozyme release from native silk films was similar to HRP, with aninitial burst of about 30%, a lag phase of 1 day and a continuousrelease between days 3 and 8 (FIG. 5B). In contrast to HRP, Lys loadedand methanol treated films did not release substantial amounts over time(FIG. 5B).

The adsorption of HRP and Lys to native film surfaces was similar forboth proteins, but apparent and statistically insignificant (p=0.08)differences were observed for methanol treated films for Lys loadedfilms but not for HRP loaded films (FIGS. 5C and 5D).

Conclusion: Drug release from silk films was a function of the drugsmolecular weight and film treatment with methanol. Sustained releaseprofiles with a linear release of bioactive protein were observed forHRP and Lys, resulting in nearly zero order kinetics from days 3 to 8(water treated films), whereas substantially less protein-drug activitywas observed upon methanol treatment. This decrease of activity wascorrelated to methanol sensitivity of Lys (and to a lesser extent forHRP). Alternatively, crystallinity can be induced by water vaportreatment (data not shown) of drug loaded films at 25° C. over asaturated Na₂SO₄ solution for 24 hours. No loss of protein activity (Lysor HRP) is expected under these vapor conditions an assumptioncorroborated by preliminary findings.

TABLE 1 Groups and treatments I 0+ I + 20 I + 90 I − 90 II + 0 II + 20II + 90 II − 90 FD4 FD4 FD4 FD4 FD4 FD4 FD4 FD4 added to preincubatedpreincubated preincubated added to preincubated preincubatedpreincubated silk-fibroin with silk- with silk- with silk- silk- withsilk- with silk- with silk- film, US fibroin fibroin fibroin fibroinfibroin fibroin fibroin treatment of solution, no solution, 20%solution, film, solution, US solution, US solution, US mixture, 90% MeOHMeOH 90% MeOH 90% treatment of treatment of treatment of MeOH treatmenttreatment treatment MeOH mixture, no mixture, mixture, treatment (n = 3)(n = 3) (n = 15) treatment MeOH 20% MeOH 90% MeOH (n = 3) (n = 3)treatment treatment treatment (n = 3) (n = 3) (n = 9)

The invention claimed is:
 1. A pharmaceutical formulation comprising afirst silk fibroin article and a second silk fibroin article, the firstsilk fibroin article comprising silk fibroin and a first therapeuticagent, the silk fibroin having a first induced change in β-sheetcrystallinity, wherein the first induced change in β-sheet crystallinityis at least equivalent to the amount of β-sheet crystallinity induced bycontacting the first silk fibroin article with 90% methanol for 3 hours,and the second silk fibroin article comprising silk fibroin and having achange in β-sheet crystallinity that is different from the silk fibroinin the first silk fibroin article, wherein the formulation exhibitscontrolled release of the first therapeutic agent over a period of about2 to 90 days.
 2. The formulation of claim 1, wherein the second silkfibroin article comprises a second therapeutic agent.
 3. The formulationof claim 2, wherein the first induced change in β-sheet crystallinity inthe silk fibroin controls release of the first therapeutic agent and/orthe second therapeutic agent.
 4. The formulation of claim 1, wherein thefirst silk fibroin article differs from the second silk fibroin articlein at least one material property.
 5. The formulation of claim 4,wherein the at least one material property is selected from the groupconsisting of silk fibroin concentration, type of therapeutic agent,concentration of therapeutic agent, dimension of the silk fibroinarticles and combinations thereof.
 6. The formulation of claim 5,wherein the therapeutic agent is selected from the group consisting ofproteins, peptides, nucleic acids, peptide nucleic acids (PNAs),aptamers, antibodies, small molecules, and any combinations thereof. 7.The formulation of claim 5, wherein the therapeutic agent is selectedfrom the group consisting of antibiotics, antiviral agents,chemotherapeutic agents, anti-rejection agents; analgesics and analgesiccombinations, anti-inflammatory agents, hormones, growth factors,antiangiogenic proteins, naturally derived or genetically engineeredproteins, polysaccharides, glycoproteins, lipoproteins, molecularcompounds, pharmacological materials, vitamins, sedatives, steroids,hypnotics, prostaglandins, radiopharmaceuticals, proteins, peptides,nucleotides, carbohydrates, simple sugars, cells, genes,anti-thrombotics, anti-metabolics, growth factor inhibitors, growthpromoters, anticoagulants, antimitotics, fibrinolytics,anti-inflammatory steroids, monoclonal antibodies, targeting ligands,and any combinations thereof.
 8. The formulation of claim 1, wherein thefirst silk fibroin article and second silk fibroin article is selectedfrom the group consisting of a thread, fiber, film, foam, mesh,hydrogel, three-dimensional scaffold, yarn, fabric, microsphere, and acoating.
 9. The formulation of claim 1, wherein the first silk fibroinarticle is dispersed in the second silk fibroin article.
 10. Theformulation of claim 9, wherein the second silk fibroin article is asilk fibroin hydrogel.
 11. The formulation of claim 1, wherein the firstsilk fibroin article and the second silk fibroin article form anonion-like structure.
 12. The formulation of claim 1, wherein change inβ-sheet crystallinity of the first silk fibroin article, the second silkfibroin article or the first silk fibroin article and the second silkfibroin article is an increase in β-sheet crystallinity.
 13. Theformulation of claim 12, wherein the increase in β-sheet crystallinitycomprises an amide II bond shift from 1540 cm⁻¹ to 1535 cm⁻¹ as observedby FTIR analysis.
 14. The formulation of claim 1, wherein the firstinduced change in β-sheet crystallinity results in an alteration in therate of release of the first therapeutic agent as compared to the rateof release from a first silk fibroin article without a first inducedchange in β-sheet crystallinity.
 15. The formulation of claim 14,wherein the alteration in the rate of release comprises absence of aninitial burst release.
 16. The formulation of claim 14, wherein thealteration in the rate of release comprises delay of an initial burstrelease.
 17. The formulation of claim 14, wherein the alteration in therate of release comprises continuous release over multiple days.
 18. Theformulation of claim 14, wherein the alteration in the rate of releasecomprises linear release over multiple days.